Nucleic acids encoding anti-IL-12 antibody

ABSTRACT

Isolated anti-IL-12 antibodies, nucleic acids encoding antibodies or antibody portions, vectors, host cells, and methods of making are useful for production of antibody or portions for treating and/or diagnosing IL-12 related conditions, diseases, and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/500,120, filed 9 Jul. 2009, now U.S. Pat. No. 7,887,807 currentlyallowed, which is a divisional of Ser. No. 11/668,256, filed 29 Jan.2007, now U.S. Pat. No. 7,560,247, issued 14 Jul. 2009, which is adivisional of U.S. application Ser. No. 10/975,883, filed Oct. 28, 2004,now U.S. Pat. No. 7,279,157, issued 9 Oct. 2007, which is a divisionalof U.S. application Ser. No. 09/920,262, filed Aug. 1, 2001, now U.S.Pat. No. 6,902,734, issued Jun. 7, 2005, which claims priority to U.S.Provisional Application Nos. 60/223,358 filed Aug. 7, 2000 and60/236,827 filed Sep. 29, 2000, each of which is entirely incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies, including specifiedportions or variants, specific for at least one Interleukin-12 (IL-12)protein or fragment thereof, as well as nucleic acids encoding suchanti-IL-12 antibodies, complementary nucleic acids, vectors, host cells,and methods of making and using thereof, including therapeuticformulations, administration and devices.

2. Related Art

Interleukin-12 (IL-12) is a heterodimeric cytokine consisting ofglycosylated polypeptide chains of 35 and 40 kD which are disulfidebonded. The cytokine is synthesized and secreted by antigen presentingcells including dendritic cells, monocytes, macrophages, B cells,Langerhans cells and keratinocytes as well as natural killer (NK) cells.IL-12 mediates a variety of biological processes and has been referredto as NK cell stimulatory factor (NKSF), T-cell stimulating factor,cytotoxic T-lymphocyte maturation factor and EBV-transformed B-cell linefactor (Curfs, J. H. A. J., et al., Clinical Microbiology Reviews,10:742-780 (1997)).

Interleukin-12 can bind to the IL-12 receptor expressed on the plasmamembrane of cells (e.g., T cells, NK cell), thereby altering (e.g.,initiating, preventing) biological processes. For example, the bindingof IL-12 to the IL-12 receptor can stimulate the proliferation ofpre-activated T cells and NK cells, enhance the cytolytic activity ofcytotoxic T cells (CTL), NK cells and LAK (lymphokine activated killer)cells, induce production of gamma interferon (IFN GAMMA) by T cells andNK cells and induce differentiation of naive Th0 cells into Th1 cellsthat produce IFN GAMMA and IL-2 (Trinchieri, G., Annual Review ofImmunology, 13:251-276 (1995)). In particular, IL-12 is vital for thegeneration of cytolytic cells (e.g., NK, CTL) and for mounting acellular immune response (e.g., a Th1 cell mediated immune response).Thus, IL-12 is critically important in the generation and regulation ofboth protective immunity (e.g., eradication of infections) andpathological immune responses (e.g., autoimmunity) (Hendrzak, J. A. andBrunda, M. J., Laboratory Investigation, 72:619-637 (1995)).Accordingly, an immune response (e.g., protective or pathogenic) can beenhanced, suppressed or prevented by manipulation of the biologicalactivity of IL-12 in vivo, for example, by means of an antibody.

Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/ormonoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestionor fusion protein products thereof) are potential therapeutic agentsthat are being investigated in some cases to attempt to treat certaindiseases. However, such antibodies or fragments can elicit an immuneresponse when administered to humans. Such an immune response can resultin an immune complex-mediated clearance of the antibodies or fragmentsfrom the circulation, and make repeated administration unsuitable fortherapy, thereby reducing the therapeutic benefit to the patient andlimiting the readministration of the antibody or fragment. For example,repeated administration of antibodies or fragments comprising non-humanportions can lead to serum sickness and/or anaphalaxis. In order toavoid these and other problems, a number of approaches have been takento reduce the immunogenicity of such antibodies and portions thereof,including chimerization and humanization, as well known in the art.These and other approaches, however, still can result in antibodies orfragments having some immunogenicity, low affinity, low avidity, or withproblems in cell culture, scale up, production, and/or low yields. Thus,such antibodies or fragments can be less than ideally suited formanufacture or use as therapeutic proteins.

Accordingly, there is a need to provide anti-IL-12 antibodies orfragments that overcome one more of these problems, as well asimprovements over known antibodies or fragments thereof.

SUMMARY OF THE INVENTION

The present invention provides isolated human, primate, rodent,mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies,immunoglobulins, cleavage products and other specified portions andvariants thereof, as well as anti-IL-12 antibody compositions, encodingor complementary nucleic acids, vectors, host cells, compositions,formulations, devices, transgenic animals, transgenic plants, andmethods of making and using thereof, as described and enabled herein, incombination with what is known in the art.

The present invention also provides at least one isolated anti-IL-12antibody as described herein. An antibody according to the presentinvention includes any protein or peptide containing molecule thatcomprises at least a portion of an immunoglobulin molecule, such as butnot limited to at least one complementarity determining region (CDR) ofa heavy or light chain or a ligand binding portion thereof, a heavychain or light chain variable region, a heavy chain or light chainconstant region, a framework region, or any portion thereof, that can beincorporated into an antibody of the present invention. An antibody ofthe invention can include or be derived from any mammal, such as but notlimited to a human, a mouse, a rabbit, a rat, a rodent, a primate, orany combination thereof, and the like.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding specific anti-IL-12 antibodies, comprising at least onespecified sequence, domain, portion or variant thereof. The presentinvention further provides recombinant vectors comprising saidanti-IL-12 antibody nucleic acid molecules, host cells containing suchnucleic acids and/or recombinant vectors, as well as methods of makingand/or using such antibody nucleic acids, vectors and/or host cells.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one IL-12 protein, subunit, fragment,portion or any combination thereof. At least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofsaid protein, which epitope is preferably comprised of at least 1-5amino acids of at least one portion thereof, such as, but not limitedto, at least one functional, extracellular, soluble, hydrophilic,external or cytoplasmic domain of said protein, or any portion thereof.

At least one antibody can optionally comprise at least one specifiedportion of at least one complementarity determining region (CDR) (e.g.,CDR1, CDR2 or CDR3 of the heavy or light chain variable region) and/orat least one constant or variable framework region or any portionthereof. At least one antibody amino acid sequence can furtheroptionally comprise at least one specified substitution, insertion ordeletion as described herein or as known in the art.

The present invention also provides at least one isolated anti-IL-12antibody as described herein, wherein the antibody has at least oneactivity, such as, but not limited to: (i) inhibition of IL-12 inducedIFN-gamma secretion; (ii) inhibition of LAK cell cytotoxicity; (iii)inhibition of IFN gamma mRNA transription; (iv) inhibition ofintracellular IFN gamma CD3+ cells; and/or (v) CD95 expression. See,e.g., Chan, et al., (1992). J. Immunol. 148 (1): 92-98; Chan, et al.,(1991). J. Exp. Med. 173 (4): 869-79; Chehimi, et al., (1992) J. Exp.Med. 175 (3): 789-96; Medvedev, et al., (1997) Cytokine 9 (6): 394-404.An anti-IL-12 antibody can thus be screened for a corresponding activityaccording to known methods, such as, but not limited to at least onebiological activity towards a IL-12 protein.

The present invention further provides at least one IL-12 anti-idiotypeantibody to at least one IL-12 antibody of the present invention. Theanti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulinmolecule, such as, but not limited to at least one complementaritydetermining region (CDR) of a heavy or light chain or a ligand bindingportion thereof, a heavy chain or light chain variable region, a heavychain or light chain constant region, a framework region, or any portionthereof, that can be incorporated into an antibody of the presentinvention. An antibody of the invention can include or be derived fromany mammal, such as but not limited to a human, a mouse, a rabbit, arat, a rodent, a primate, and the like.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding at least one IL-12 anti-idiotype antibody, comprising at leastone specified sequence, domain, portion or variant thereof. The presentinvention further provides recombinant vectors comprising said IL-12anti-idiotype antibody encoding nucleic acid molecules, host cellscontaining such nucleic acids and/or recombinant vectors, as well asmethods of making and/or using such anti-idiotype antibody nucleicacids, vectors and/or host cells.

The present invention also provides at least one method for expressingat least one anti-IL-12 antibody, or IL-12 anti-idiotype antibody, in ahost cell, comprising culturing a host cell as described herein underconditions wherein at least one anti-IL-12 antibody is expressed indetectable and/or recoverable amounts.

The present invention also provides at least one composition comprising(a) an isolated anti-IL-12 antibody encoding nucleic acid and/orantibody as described herein; and (b) a suitable carrier or diluent. Thecarrier or diluent can optionally be pharmaceutically acceptable,according to known carriers or diluents. The composition can optionallyfurther comprise at least one further compound, protein or composition.

The present invention further provides at least one anti-IL-12 antibodymethod or composition, for administering a therapeutically effectiveamount to modulate or treat at least one IL-12 related condition in acell, tissue, organ, animal or patient and/or, prior to, subsequent to,or during a related condition, as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery of a therapeutically or prophylacticallyeffective amount of at least one anti-IL-12 antibody, according to thepresent invention.

The present invention further provides at least one anti-IL-12 antibodymethod or composition, for diagnosing at least one IL-12 relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery for diagnosing of at least one anti-IL-12antibody, according to the present invention.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are graphs showing concentration-dependent binding ofhuman anti-IL-12 mAbs to immobilized human IL-12. Anti-IL-12 antibodieswere serially diluted in 1% BSA/PBS and incubated on rhIL-12 coatedplates for 1 hour at 37° C. Plates were washed twice with 0.02% Tween 20(polyoxyethylene(20) sorbitan monolaurate), 0.15M saline and then probedwith horse radish peroxidase (HRP) labeled goat anti-human IgG kappaspecific antibody for 1 hour at room temperature. Plates were againwashed, developed with o-phenylenediamine (OPD) substrate and theoptical density (OD) of each well was measured at 490 nm.

FIG. 2: Lanes from left to right in Figures A and B contain human IL-12,human IL-12 p40, murine IL-12, and prestained molecular weight markers.FIG. 2A shows bands stained from total protein. The primary bands ineach lane are human IL-12 (75 kd), p40 human IL-12 (40 kd), and murineIL-12 (75 kd). FIG. 2B shows a western blot prepared from a gelidentical to that shown in FIG. 2A. Blot was reacted with C340 followedby HRP labeled goat anti-human IgG and specifically detected human IL-12(monomer and multimers) and human IL-12 p40 only. A control blot (notshown) reacted with HRP labeled goat anti-human IgG did not display anybands.

FIG. 3: Reverse transcription-PCR analysis of IFNγ gene expression inhuman PBL's treated with IL-2, IL-12, IL-2+IL-12 with and withoutanti-IL-12 antibody C340, 8.6.2, isotype control antibody. Total RNA wasreverse transcribed, amplified by PCR using gene-specific primers. Thelevel of β-actin mRNA in each sample was also determined, which servedas a control for mRNA integrity and content.

FIG. 4 is a histogram showing that human anti-IL-12 mAb (C340) inhibitsproduction of interferon-γ (IFNγ) by monocyte depleted CD3+ peripheralblood mononuclear cells (PBMC) stimulated with IL-2 plus IL-12. PBMCwere cultured for five hours in control media (no added cytokines),media supplemented with IL-12 (0.1 ng/ml) plus IL-2 (50 IU/ml)(IL-12/IL-2), control media containing mAb C340 (10 μg/ml) andIL-12/IL-2 media containing mAb C340 (10 μg/ml). Intracellular IFNγ wasmeasured by two color immunostaining with CD3-PE and IFNγ-FITC. Data areshown for one donor.

FIG. 5 is a graph showing dose-dependent inhibition of IFNγ secretion byIL-2 plus IL-12 stimulated peripheral blood lymphocytes with twodifferent lots of a human anti-IL-12 mAb (C340). Human PBL (8×106/ml)were cultured for 24 hours with 10 U/ml IL-2, IL-2 plus 400 pg/ml IL-12,or IL-2 plus IL-12 and mAb C340 as indicated. The culture supernatentswere removed and assayed for IFNγ by EIA.

FIG. 6 is a histogram showing dose-dependent inhibition of IL-12 plusIL-2 induced LAK cell cytotoxicity by a human anti-IL-12 mAb (C340). LAKeffector cells (human PBL, 8×106/ml) were cultured for 24 hours withIL-12 (400 pg/ml) plus IL-2 (10 U/ml) and mAb C340 (5000 ng/ml or 50ng/ml as indicated). The LAK effector cells were washed and culturedwith 51Cr labeled Raji target cells for four hours at an effector totarget (E:T) ration of 80:1, and the quantity of 51Cr released into themedia upon Raji cell lysis was measured. Results are expressed as themean of three normal donors standard error. IL-12 positive control(IL-12) is effector cells incubated with IL-12 and without antibody.Background (BKGD) is effector cells incubated without IL-12 or antibody.

FIGS. 7A and 7B are histograms showing that IL-12 plus IL-2-inducedexpression of CD95 on CD3+ peripheral blood mononuclear cells isinhibited by human anti-IL-12 mAb (C340). PBMC were cultured for 72hours in media containing 0.1 ng/ml IL-12 and a suboptimal dose of IL-2(50 IU/ml) in the presence or absence of mAb C340 (10 μg/ml). CD95expression was measured by flow cytometry of cells stained withanti-CD95-FITC. Gating was performed using two-color analysis (CD3 orCD56-PE vs. CD95-FITC) and forward vs. orthogonal light scatter.

FIG. 8 is a graph showing that recombinant human anti-human IL-12antibodies (rC340) bind to immobilized IL-12 in a manner that isindistinguishable from purified mAb C340. The concentration of rC340 inthe supernatants of three rC340-producing recombinant cell lines wasdetermined, and the supernatants were evaluated for IL-12 binding in anELISA. Plates were coated with 2 μg/ml human IL-12 and incubated withpurified mAb C340 from the original hybridoma (standard) or thesupernatants of recombinant cell lines. IL-12-bound antibody wasdetected using alkaline phosphatase-conjugated goat anti-human IgG(heavy chain+light chain).

FIGS. 9A-9C are graphs showing growth kinetics and the quantity ofantibody secreted by three independently-derived rC340-producingrecombinant cell subclones (FIG. 9A, subclone C379B; FIG. 9B, subcloneC381A; FIG. 9C, subclone C389A). Recombinant cells were seeded into T75flasks at a starting density of 2×10⁵ cells/ml in standard media. Atvarious times, cells were resuspended and the number of live cells andthe quantity (μg/ml) of rC340 in the media were determined.

DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticanti-IL-12 human, primate, rodent, mammalian, chimeric, humanized orCDR-grafted, antibodies and IL-12 anti-idiotype antibodies thereto, aswell as compositions and encoding nucleic acid molecules comprising atleast one polynucleotide encoding at least one anti-IL-12 antibody oranti-idiotype antibody. The present invention further includes, but isnot limited to, methods of making and using such nucleic acids andantibodies and anti-idiotype antibodies, including diagnostic andtherapeutic compositions, methods and devices.

As used herein, an “anti-Interleukin-12 antibody,” “anti-IL-12antibody,” “anti-IL-12 antibody portion,” or “anti-IL-12 antibodyfragment” and/or “anti-IL-12 antibody variant” and the like include anyprotein or peptide containing a molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework region, or any portion thereof, or at least one portion of anIL-12 receptor or binding protein, which can be incorporated into anantibody of the present invention. Such antibody optionally furtheraffects a specific ligand, such as but not limited to where suchantibody modulates, decreases, increases, antagonizes, agonizes,mitigates, alleviates, blocks, inhibits, abrogates and/or interfereswith at least one IL-12 activity or binding, or with IL-12 receptoractivity or binding, in vitro, in situ and/or in vivo. As a non-limitingexample, a suitable anti-IL-12 antibody, specified portion, or variantof the present invention can bind at least one IL-12, or specifiedportions, variants or domains thereof. A suitable anti-IL-12 antibody,specified portion, or variant can also optionally affect at least oneIL-12 activity or function, such as but not limited to, RNA, DNA orprotein synthesis, IL-12 release, IL-12 receptor signaling, membraneIL-12 cleavage, IL-12 activity, IL-12 production and/or synthesis. Theterm “antibody” is further intended to encompass antibodies, digestionfragments, specified portions and variants thereof, including antibodymimetics or comprising portions of antibodies that mimic the structureand/or function of an antibody or specified fragment or portion thereof,including single chain antibodies and fragments thereof. Functionalfragments include antigen-binding fragments that bind to a mammalianIL-12. For example, antibody fragments capable of binding to IL-12 orportions thereof, including, but not limited to Fab (e.g., by papaindigestion), Fab′ (e.g., by pepsin digestion and partial reduction) andF(ab′)₂ (e.g., by pepsin digestion), facb (e.g., by plasmin digestion),pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsindigestion, partial reduction and reaggregation), Fv or scFv (e.g., bymolecular biology techniques) fragments, are encompassed by theinvention (see, e.g., Colligan, Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) issubstantially non-immunogenic in humans, with only minor sequencechanges or variations. Similarly, antibodies designated primate (monkey,baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig,hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one IL-12 protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

Anti-IL-12 antibodies (also termed IL-12 antibodies) useful in themethods and compositions of the present invention can optionally becharacterized by high affinity binding to IL-12 and optionally andpreferably having low toxicity. In particular, an antibody, specifiedfragment or variant of the invention, where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively, optionally and preferably, possess lowimmunogenicity, is useful in the present invention. The antibodies thatcan be used in the invention are optionally characterized by theirability to treat patients for extended periods with measurablealleviation of symptoms and low and/or acceptable toxicity. Low oracceptable immunogenicity and/or high affinity, as well as othersuitable properties, can contribute to the therapeutic results achieved.“Low immunogenicity” is defined herein as raising significant HAHA, HACAor HAMA responses in less than about 75%, or preferably less than about50% of the patients treated and/or raising low titres in the patienttreated (less than about 300, preferably less than about 100 measuredwith a double antigen enzyme immunoassay) (see, e.g., Elliott et al.,Lancet 344:1125-1127 (1994)), entirely incorporated herein by reference.

Utility

The isolated nucleic acids of the present invention can be used forproduction of at least one anti-IL-12 antibody or specified variantthereof, which can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one IL-12 condition, selected from, but notlimited to, at least one of an immune disorder or disease, acardiovascular disorder or disease, an infectious, malignant, and/orneurologic disorder or disease, or other known or specified IL-12related condition.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts.

CITATIONS

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. The followingreferences are entirely incorporated herein by reference: Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlowand Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.(1989); Colligan, et al., eds., Current Protocols in Immunology, JohnWiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocolsin Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

Antibodies of the Present Invention

At least one anti-IL-12 antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual,Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

Human antibodies that are specific for human IL-12 proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas an isolated and/or IL-12 protein or a portion thereof (includingsynthetic molecules, such as synthetic peptides). Other specific orgeneral mammalian antibodies can be similarly raised. Preparation ofimmunogenic antigens, and monoclonal antibody production can beperformed using any suitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, bovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like), display library; (e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys). See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/U594/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins; U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME)), each entirelyincorporated herein by reference, or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference, as well as related patents and applicationthat are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (USPat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V.,Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/researchtools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/˜mrc7/mikeimages.html; www.antibodyresource.com;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/˜hcenter/index.html; www.biotech.ufl.edu/˜hcl/;www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com/table.asp;www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/˜fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/˜rek/AEPStart.html;baserv.uci.kun.nl/˜jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/virN_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/˜martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/˜honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/˜ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html.Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.Generally part or all of the non-human or human CDR sequences aremaintained while the non-human sequences of the variable and constantregions are replaced with human or other amino acids. Antibodies canalso optionally be humanized with retention of high affinity for theantigen and other favorable biological properties. To achieve this goal,humanized antibodies can be optionally prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

The anti-IL-12 antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, hamster, non-human primate,and the like) capable of producing a repertoire of human antibodies, asdescribed herein and/or as known in the art. Cells that produce a humananti-IL-12 antibody can be isolated from such animals and immortalizedusing suitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol. 6(4) 579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90 (8) 3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93 (1995) and Fishwald et al., Nat Biotechnol 14 (7):845-851(1996), which are each entirely incorporated herein by reference).Generally, these mice comprise at least one transgene comprising DNAfrom at least one human immunoglobulin locus that is functionallyrearranged, or which can undergo functional rearrangement. Theendogenous immunoglobulin loci in such mice can be disrupted or deletedto eliminate the capacity of the animal to produce antibodies encoded byendogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge Antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456, assigned to Enzon; 5,223,409, 5,403,484, 5,571,698,5,837,500, assigned to Dyax, 5,427,908, 5,580,717, assigned to Affymax;5,885,793, assigned to Cambridge antibody Technologies; 5,750,373,assigned to Genentech, 5,618,920, 5,595,898, 5,576,195, 5,698,435,5,693,493, 5,698,417, assigned to Xoma, Colligan, supra; Ausubel, supra;or Sambrook, supra, each of the above patents and publications entirelyincorporated herein by reference.

Antibodies of the present invention can also be prepared using at leastone anti-IL-12 antibody encoding nucleic acid to be provided totransgenic animals or mammals, such as goats, cows, horses, sheep, andthe like, that produce such antibodies in their milk. Such antibodiescan be provided using known methods. See, e.g., but not limited to, U.S.Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616;5,565,362; 5,304,489, and the like, each of which is entirelyincorporated herein by reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-IL-12 antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to known methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. Each of the abovereferences is entirely incorporated herein by reference.

The antibodies of the invention can bind human IL-12 with a wide rangeof affinities (K_(D)). In a preferred embodiment, at least one human mAbof the present invention can optionally bind human IL-12 with highaffinity. For example, a human mAb can bind human IL-12 with a K_(D)equal to or less than about 10⁻⁷ M, such as but not limited to, 0.1-9.9(or any range or value therein)×10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²,10⁻¹³ or any range or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, specified fragments, variants orconsensus sequences thereof, or a deposited vector comprising at leastone of these sequences, a nucleic acid molecule of the present inventionencoding at least one anti-IL-12 antibody can be obtained using methodsdescribed herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-IL-12 antibody or variable region (e.g., SEQ ID NOS:7, 8);and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-IL-12antibody as described herein and/or as known in the art. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-IL-12 antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules of the present invention include a nucleic acidencoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LCCDR3, HC variable region and LC variable region.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-IL-12 antibody caninclude, but are not limited to, those encoding the amino acid sequenceof an antibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample, ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence, is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 toInnis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 toGyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, etal; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al, with the tradename NASBA), the entire contents of whichare incorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-IL-12 antibody by recombinant techniques, as iswell known in the art. See, e.g., Sambrook, et al., supra; Ausubel, etal., supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017),ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). Preferred host cells include cells of lymphoidorigin such as myeloma and lymphoma cells. Particularly preferred hostcells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) andSP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularlypreferred embodiment, the recombinant cell is a P3X63Ab8.653 or aSP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Antibody

An anti-IL-12 antibody can be recovered and purified from recombinantcell cultures by well-known methods including, but not limited to,protein A purification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Anti-IL-12 Antibodies

The isolated antibodies of the present invention comprise an antibodyencoded by any one of the polynucleotides of the present invention asdiscussed more fully herein, or any isolated or prepared antibody.Preferably, the human antibody or antigen-binding fragment binds humanIL-12 and, thereby partially or substantially neutralizes at least onebiological activity of the protein. An antibody, or specified portion orvariant thereof, that partially or preferably substantially neutralizesat least one biological activity of at least one IL-12 protein orfragment can bind the protein or fragment and thereby inhibit activitiesmediated through the binding of IL-12 to the IL-12 receptor or throughother IL-12-dependent or mediated mechanisms. As used herein, the term“neutralizing antibody” refers to an antibody that can inhibit anIL-12-dependent activity by about 20-120%, preferably by at least about10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100% or more depending on the assay. The capacity of ananti-IL-12 antibody to inhibit an IL-12-dependent activity is preferablyassessed by at least one suitable IL-12 protein or receptor assay, asdescribed herein and/or as known in the art. A human antibody of theinvention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotypeand can comprise a kappa or lambda light chain. In one embodiment, thehuman antibody comprises an IgG heavy chain or defined fragment, forexample, at least one of isotypes, IgG1, IgG2, IgG3 or IgG4. Antibodiesof this type can be prepared by employing a transgenic mouse or othertrangenic non-human mammal comprising at least one human light chain(e.g., IgG, IgA and IgM (e.g., γ1, γ2, γ3, γ4) transgenes as describedherein and/or as known in the art). In another embodiment, theanti-human IL-12 human antibody comprises an IgG1 heavy chain and anIgG1 light chain.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one IL-12 protein, subunit, fragment,portion or any combination thereof. At least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofsaid protein, which epitope is preferably comprised of at least oneextracellular, soluble, hydrophilic, external or cytoplasmic portion ofsaid protein. At least one specified epitope can comprise anycombination of at least one amino acid sequence of at least 1-3 aminoacids to the entire specified portion of contiguous amino acids of theSEQ ID NO:9.

Generally, the human antibody or antigen-binding fragment of the presentinvention will comprise an antigen-binding region that comprises atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one heavy chain variable region and at least onehuman complementarity determining region (CDR1, CDR2 and CDR3) orvariant of at least one light chain variable region. As a non-limitingexample, the antibody or antigen-binding portion or variant can compriseat least one of the heavy chain CDR3 having the amino acid sequence ofSEQ ID NO:3, and/or a light chain CDR3 having the amino acid sequence ofSEQ ID NO:6. In a particular embodiment, the antibody or antigen-bindingfragment can have an antigen-binding region that comprises at least aportion of at least one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NOS:1, 2, and/or 3). In another particular embodiment, theantibody or antigen-binding portion or variant can have anantigen-binding region that comprises at least a portion of at least onelight chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 4,5, and/or 6). In a preferred embodiment the three heavy chain CDRs andthe three light chain CDRs of the antibody or antigen-binding fragmenthave the amino acid sequence of the corresponding CDR of at least one ofmAb 12B75, C340, or any others as described herein. Such antibodies canbe prepared by chemically joining together the various portions (e.g.,CDRs, framework) of the antibody using conventional techniques, bypreparing and expressing a (i.e., one or more) nucleic acid moleculethat encodes the antibody using conventional techniques of recombinantDNA technology or by using any other suitable method.

The anti-IL-12 antibody can comprise at least one of a heavy or lightchain variable region having a defined amino acid sequence. For example,in a preferred embodiment, the anti-IL-12 antibody comprises at leastone of at least one heavy chain variable region, optionally having theamino acid sequence of SEQ ID NO:7 and/or at least one light chainvariable region, optionally having the amino acid sequence of SEQ IDNO:8. Antibodies that bind to human IL-12 and that comprise a definedheavy or light chain variable region can be prepared using suitablemethods, such as phage display (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868 (1998)) or methods that employ transgenic animals, as knownin the art and/or as described herein. For example, a transgenic mouse,comprising a functionally rearranged human immunoglobulin heavy chaintransgene and a transgene comprising DNA from a human immunoglobulinlight chain locus that can undergo functional rearrangement, can beimmunized with human IL-12 or a fragment thereof to elicit theproduction of antibodies. If desired, the antibody producing cells canbe isolated and hybridomas or other immortalized antibody-producingcells can be prepared as described herein and/or as known in the art.Alternatively, the antibody, specified portion or variant can beexpressed using the encoding nucleic acid or portion thereof in asuitable host cell.

The invention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human IL-12 with high affinity(e.g., K_(D) less than or equal to about 10⁻⁹ M) Amino acid sequencesthat are substantially the same as the sequences described hereininclude sequences comprising conservative amino acid substitutions, aswell as amino acid deletions and/or insertions. A conservative aminoacid substitution refers to the replacement of a first amino acid by asecond amino acid that has chemical and/or physical properties (e.g.,charge, structure, polarity, hydrophobicity/hydrophilicity) that aresimilar to those of the first amino acid. Conservative substitutionsinclude replacement of one amino acid by another within the followinggroups: lysine (K), arginine (R) and histidine (H), aspartate (D) andglutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T),tyrosine (Y), K, R, H, D and E, alanine (A), valine (V), leucine (L),isoleucine (I), proline (P), phenylalanine (F), tryptophan (W),methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino Acid Codes

The amino acids that make up anti-IL-12 antibodies of the presentinvention are often abbreviated. The amino acid designations can beindicated by designating the amino acid by its single letter code, itsthree letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts, B., et al., Molecular Biology of TheCell, Third Ed., Garland Publishing, Inc., New York, 1994):

SINGLE THREE LETTER LETTER THREE NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Asparticacid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU GGly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile IsoleucineAUA, AUC, AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC,CUG, CUU M Met Methionine AUG N Asn Asparagine AAC, AAU P Pro ProlineCCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG,CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T ThrThreonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W TrpTryptophan UGG Y Tyr Tyrosine UAC, UAU

An anti-IL-12 antibody of the present invention can include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-IL-12 Ig-derived protein, fragment orvariant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range orvalue therein, as specified herein.

Amino acids in an anti-IL-12 antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one IL-12 neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

Anti-IL-12 antibodies of the present invention can include, but are notlimited to, at least one portion, sequence or combination selected from5 to all of the contiguous amino acids of at least one of SEQ ID NOS:1,2, 3, 4, 5, 6.

IL-12 antibodies or specified portions or variants of the presentinvention can include, but are not limited to, at least one portion,sequence or combination selected from at least 3-5 contiguous aminoacids of SEQ ID NO:1, 5-17 contiguous amino acids of SEQ ID NO:2, 5-10contiguous amino acids of SEQ ID NO:3, 5-11 contiguous amino acids ofSEQ ID NO:4, 5-7 contiguous amino acids of SEQ ID NO:5; 5-9 contiguousamino acids of SEQ ID NO:6; Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7.

An anti-IL-12 antibody can further optionally comprise a polypeptide ofat least one of 70-100% of 5, 17, 10, 11, 7, 9, 119, or 108 contiguousamino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7 or 8.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7 or 8. For example,the amino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:3. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 7 and 8. The antibodies of the presentinvention, or specified variants thereof, can comprise any number ofcontiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10-100% of the number of contiguous residues in ananti-IL-12 antibody. Optionally, this subsequence of contiguous aminoacids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ormore amino acids in length, or any range or value therein. Further, thenumber of such subsequences can be any integer selected from the groupconsisting of from 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24 (1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56 (4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

Anti-Idiotype Antibodies to Anti-IL-12 IG Derived Protein Compositions

In addition to monoclonal or chimeric anti-IL-12 antibodies, the presentinvention is also directed to an anti-idiotypic (anti-Id) antibodyspecific for such antibodies of the invention. An anti-Id antibody is anantibody which recognizes unique determinants generally associated withthe antigen-binding region of another antibody. The anti-Id can beprepared by immunizing an animal of the same species and genetic type(e.g. mouse strain) as the source of the Id antibody with the antibodyor a CDR containing region thereof. The immunized animal will recognizeand respond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

Anti-IL-12 IG Derived Protein Compositions

The present invention also provides at least one anti-IL-12 antibodycomposition comprising at least one, at least two, at least three, atleast four, at least five, at least six or more anti-IL-12 antibodiesthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcompositions comprise non-naturally occurring compositions comprising atleast one or two full length, C- and/or N-terminally deleted variants,domains, fragments, or specified variants, of the anti-IL-12 antibodyamino acid sequence selected from the group consisting of 70-100% of thecontiguous amino acids of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7 or 8, orspecified fragments, domains or variants thereof. Preferred anti-IL-12derived protein, fragment or variant compositions include at least oneor two full length, fragments, domains or variants as at least one CDRcontaining portions of the anti-IL-12 antibody sequence of 70-100% ofSEQ ID NOS: 1, 2, 3, 4, 5, 6, or specified fragments, domains orvariants thereof. Further preferred compositions comprise 40-99% of atleast one of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or specifiedfragments, domains or variants thereof. Such composition percentages areby weight, volume, concentration, molarity, or molality as liquid or drysolutions, mixtures, suspension, emulsions or colloids, as known in theart or as described herein.

Anti-IL-12 antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy, optionally further comprisingat least one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial (e.g., an aminoglycoside, an antifungal, an antiparasitic,an antiviral, a carbapenem, cephalosporin, a fluoroquinolone, amacrolide, a penicillin, a sulfonamide, a tetracycline, anotherantimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid,a diabetes related agent, a mineral, a nutritional, a thyroid agent, avitamin, a calcium related hormone, an antidiarrheal, an antitussive, anantiemetic, an antiulcer, a laxative, an anticoagulant, anerythropoietin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF,Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, animmunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine,daclizumab), a growth hormone, a hormone replacement drug, an estrogenreceptor modulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examplesof such cytokines include, but are not limited to, any of IL-1 to IL-23.Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium difficile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophile, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-IL-12 antibody compounds, compositions or combinations of thepresent invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-IL-12 antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-IL-12 antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-IL-12 antibody compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-IL-12 antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference,”52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents.

Formulations

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one anti-IL-12 antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-IL-12 antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one anti-IL-12 antibody, anda second vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one anti-IL-12 antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

At least one anti-IL-12 antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one anti-IL-12 antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-IL-12 antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-IL-12 antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-IL-12 antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingwater, a preservative and/or excipients, preferably a phosphate bufferand/or saline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2° C. to about 40° C. and retain the biologically activity of theprotein for extended periods of time, thus, allowing a package labelindicating that the solution can be held and/or used over a period of 6,12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent isused, such label can include use up to 1-12 months, one-half, one and ahalf, and/or two years.

The solutions of at least one anti-IL-12 antibody in the invention canbe prepared by a process that comprises mixing at least one antibody inan aqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-IL-12antibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject® NovoPen®, B-D® Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen® Roferon Pen®Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute at least one anti-IL-12 antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-IL-12 antibody and a selectedbuffer, preferably a phosphate buffer containing saline or a chosensalt. Mixing at least one antibody and buffer in an aqueous diluent iscarried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one antibody in water or buffer is combined with the desiredbuffering agent in water in quantities sufficient to provide the proteinand buffer at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-IL-12 antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one anti-IL-12 antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications

The present invention also provides a method for modulating or treatingat least one immune related disease, in a cell, tissue, organ, animal,or patient, including, but not limited to, at least one of rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatorybowel disease, ulcerative colitis, systemic lupus erythematosus,antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis,idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitivity reactions, allergic rhinitis, hayfever, perennial rhinitis, conjunctivitis, endometriosis, asthma,urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,hemolytic disease, thrombocytopenia, graft rejection of any organ ortissue, kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g., theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al.,eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic atherosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aortic andperipheral aneurysms, aortic dissection, inflammation of the aorta,occlusion of the abdominal aorta and its branches, peripheral vasculardisorders, occlusive arterial disorders, peripheral atheroscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy.

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like;

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome(MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

The present invention also provides a method for modulating or treatingat least one neurologic disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi-system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit, such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16^(th) Edition,Merck & Company, Rahway, N.J. (1992)

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases, wherein theadministering of said at least one anti-IL-12 antibody, specifiedportion or variant thereof, further comprises administering, before,concurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonism. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, antigen-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signaling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/orpreferably in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

Chimeric antibody cA2 consists of the antigen binding variable region ofthe high-affinity neutralizing mouse anti-human TNFα IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric antibody cA2 and recombinant human TNFα, the affinityconstant of chimeric antibody cA2 was calculated to be 1.04×10¹⁰M⁻¹.Preferred methods for determining monoclonal antibody specificity andaffinity by competitive inhibition can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, NewYork, (1992-2000); Kozbor et al., Immunol. Today, 4:72-79 (1983);Ausubel et al., eds. Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2000); and Muller, Meth. Enzymol.,92:589-601 (1983), which references are entirely incorporated herein byreference.

In a particular embodiment, murine monoclonal antibody A2 is produced bya cell line designated c134A. Chimeric antibody cA2 is produced by acell line designated c168A.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Möller, A. et al., Cytokine 2 (3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathj en et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

TNF Receptor Molecules

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFα with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highaffinity, as well as other undefined properties, can contribute to thetherapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6 (6): 616-623 (1994); Baker et al., Eur. J.Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNFα with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

Cytokines include any known cytokine. See, e.g.,www.CopewithCytokines.com. Cytokine antagonists include, but are notlimited to, any antibody, fragment or mimetic, any soluble receptor,fragment or mimetic, any small molecule antagonist, or any combinationthereof.

Therapeutic Treatments. Any method of the present invention can comprisea method for treating an IL-12 mediated disorder, comprisingadministering an effective amount of a composition or pharmaceuticalcomposition comprising at least one anti-IL-12 antibody to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy. Such a method can optionally further compriseco-administration or combination therapy for treating such immunediseases, wherein the administering of at least one anti-IL-12 antibody,specified portion or variant thereof, further comprises administeringbefore, concurrently, and/or after, at least one selected from at leastone TNF antagonist (e.g., but not limited to a TNF antibody or fragment,a soluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-IL-12antibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one anti-IL-12 antibody per kilogramof patient per dose, and preferably from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 μg/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes of can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one anti-IL-12 antibody according to the present invention. Whilepulmonary administration is used in the following description, othermodes of administration can be used according to the present inventionwith suitable results.

IL-12 antibodies of the present invention can be delivered in a carrier,as a solution, emulsion, colloid, or suspension, or as a dry powder,using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as an aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least oneanti-IL-12 antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.At least one anti-IL-12 antibody composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably at least one anti-IL-12antibody composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses. According to theinvention, at least one anti-IL-12 antibody can be delivered by any of avariety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of antibodies are alsoknown in the art. All such devices can use of formulations suitable forthe administration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus®(Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-IL-12 antibody isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone antibody of the present invention. For example, delivery by theinhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 μm, preferably about 1-5 μm, for goodrespirability.

Administration of IL-12 Antibody Compositions as a Spray

A spray including IL-12 antibody composition protein can be produced byforcing a suspension or solution of at least one anti-IL-12 antibodythrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of at least one anti-IL-12 antibodycomposition protein delivered by a sprayer have a particle size lessthan about 10 μm, preferably in the range of about 1 μm to about 5 μm,and most preferably about 2 μm to about 3 μm.

Formulations of at least one anti-IL-12 antibody composition proteinsuitable for use with a sprayer typically include antibody compositionprotein in an aqueous solution at a concentration of about 0.1 mg toabout 100 mg of at least one anti-IL-12 antibody composition protein perml of solution or mg/gm, or any range or value therein, e.g., but notlimited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml ormg/gm. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the antibody composition protein, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating antibody composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulatingantibody composition proteins include sucrose, mannitol, lactose,trehalose, glucose, or the like. The antibody composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the antibody composition protein causedby atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as IL-12 antibodies, or specified portionsor variants, can also be included in the formulation.

Administration of IL-12 Antibody Compositions by a Nebulizer

Antibody composition protein can be administered by a nebulizer, such asjet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer,a compressed air source is used to create a high-velocity air jetthrough an orifice. As the gas expands beyond the nozzle, a low-pressureregion is created, which draws a solution of antibody compositionprotein through a capillary tube connected to a liquid reservoir. Theliquid stream from the capillary tube is sheared into unstable filamentsand droplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

Formulations of at least one anti-IL-12 antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one anti-IL-12 antibodyprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-IL-12antibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-IL-12 antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneanti-IL-12 antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. At least one anti-IL-12 antibody formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the at least one anti-IL-12 antibody caused byatomization of the solution in forming an aerosol. Various conventionalsurfactants can be employed, such as polyoxyethylene fatty acid estersand alcohols, and polyoxyethylene sorbital fatty acid esters. Amountswill generally range between 0.001 and 4% by weight of the formulation.Especially preferred surfactants for purposes of this invention arepolyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20, orthe like. Additional agents known in the art for formulation of aprotein such as antibody protein can also be included in theformulation.

Administration of IL-12 Antibody Compositions by a Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one anti-IL-12antibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably about 1 pan to about 5 μm, and most preferably about 2 μm toabout 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of an antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one anti-IL-12 antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-IL-12 antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize at least one anti-IL-12 antibody as a suspension inthe propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some casessolution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein such asprotein can also be included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one anti-IL-12 antibody compositions via devices not describedherein.

Oral Formulations and Administration

Formulations for oral rely on the co-administration of adjuvants (e.g.,resorcinols and nonionic surfactants such as polyoxyethylene oleyl etherand n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, alpha.-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,871,753 are used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods ofadministering at least one anti-IL-12 antibody include an emulsioncomprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670).Mucous surfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos.5,849,695).

Transdermal Formulations and Administration

For transdermal administration, at least one anti-IL-12 antibody isencapsulated in a delivery device such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers such as polyhydroxy acids such as polylactic acid, polyglycolicacid and copolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. Nos. 5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1 Cloning and Expression of IL-12 Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of IL-12 antibody. Plasmid pC4is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Theplasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (e.g., alpha minus MEM, LifeTechnologies, Gaithersburg, Md.) supplemented with the chemotherapeuticagent methotrexate. The amplification of the DHFR genes in cellsresistant to methotrexate (MTX) has been well documented (see, e.g., F.W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C.Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M.A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the IL-12in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete IL-12 antibody is used,corresponding to HC and LC variable regions of an IL-12 antibody of thepresent invention, according to known method steps. Isolated nucleicacid encoding a suitable human constant region (i.e., HC and LC regions)is also used in this construct (e.g., as provided in vector p1351:.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 2 Generation of High Affinity Human IgG Monoclonal AntibodiesReactive with Human IL-12 Using Transgenic Mice

Summary

Transgenic mice have been used that contain human heavy and light chainimmunoglobulin genes to generate high affinity, completely human,monoclonal antibodies that can be used therapeutically to inhibit theaction of IL-12 for the treatment of one or more IL-12-mediated disease.(CBA/J×C57/BL6/J) F₂ hybrid mice containing human variable and constantregion antibody transgenes for both heavy and light chains are immunizedwith human recombinant IL-12 (Taylor et al., Intl. Immunol. 6:579-591(1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M.,Nature Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology14:845-851 (1996)). Several fusions yielded one or more panels ofcompletely human IL-12 reactive IgG monoclonal antibodies. Thecompletely human anti-IL-12 antibodies are further characterized. Allare IgG1κ. Such antibodies are found to have affinity constantssomewhere between 1×10⁹ and 9×10¹². The unexpectedly high affinities ofthese fully human monoclonal antibodies make them suitable candidatesfor therapeutic applications in IL-12 related diseases, pathologies ordisorders.

ABBREVIATIONS

-   BSA—bovine serum albumin-   CO₂—carbon dioxide-   DMSO—dimethyl sulfoxide-   EIA—enzyme immunoassay-   FBS—fetal bovine serum-   H₂O₂—hydrogen peroxide-   HRP—horseradish peroxidase\-   ID—interadermal-   Ig—immunoglobulin-   IL-12—interleukin-12-   IP—intraperitoneal-   IV—intravenous-   Mab—monoclonal antibody-   OD—optical density-   OPD—o-Phenylenediamine dihydrochloride-   PEG—polyethylene glycol-   PSA—penicillin, streptomycin, amphotericin-   RT—room temperature-   SQ—subcutaneous-   v/v—volume per volume-   w/v—weight per volume    Materials and Methods    Animals

Transgenic mice that can express human antibodies are known in the art(and are commercially available (e.g., from GenPharm International, SanJose, Calif.; Abgenix, Freemont, Calif., and others) that express humanimmunoglobulins but not mouse IgM or IgK. For example, such transgenicmice contain human sequence transgenes that undergo V(D)J joining,heavy-chain class switching, and somatic mutation to generate arepertoire of human sequence immunoglobulins (Lonberg, et al., Nature368:856-859 (1994)). The light chain transgene can be derived, e.g., inpart from a yeast artificial chromosome clone that includes nearly halfof the germline human VK region. In addition, the heavy-chain transgenecan encode both human μ and human λ1 (Fishwild, et al., NatureBiotechnology 14:845-851 (1996)) and/or λ3 constant regions. Micederived from appropriate genotopic lineages can be used in theimmunization and fusion processes to generate fully human monoclonalantibodies to IL-12.

Immunization

One or more immunization schedules can be used to generate theanti-IL-12 human hybridomas. The first several fusions can be performedafter the following exemplary immunization protocol, but other similarknown protocols can be used. Several 14-20 week old female and/orsurgically castrated transgenic male mice are immunized IP and/or IDwith 1-1000 μg of recombinant human IL-12 emulsified with an equalvolume of TITERMAX or complete Freund's adjuvant in a final volume of100-400 μL (e.g., 200). Each mouse can also optionally receive 1-10 μgin 100 μL physiological saline at each of 2 SQ sites. The mice can thenbe immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400μg) and SQ (1-400 μg×2) with IL-12 emulsified with an equal volume ofTITERMAX or incomplete Freund's adjuvant. Mice can be bled 12-25 and25-40 days later by retro-orbital puncture without anti-coagulant. Theblood is then allowed to clot at RT for one hour and the serum iscollected and titered using an IL-12 EIA assay according to knownmethods. Fusions are performed when repeated injections do not causetiters to increase. At that time, the mice can be given a final IVbooster injection of 1-400 μg IL-12 diluted in 100 μL physiologicalsaline. Three days later, the mice can be euthanized by cervicaldislocation and the spleens removed aseptically and immersed in 10 mL ofcold phosphate buffered saline (PBS) containing 100 U/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL amphotericin B (PSA). The splenocytesare harvested by sterilely perfusing the spleen with PSA-PBS. The cellsare washed once in cold PSA-PBS, counted using Trypan blue dye exclusionand resuspended in RPMI 1640 media containing 25 mM Hepes.

Cell Fusion

Fusion can be carried out at a 1:1 to 1:10 ratio of murine myeloma cellsto viable spleen cells according to known methods, e.g., as known in theart. As a non-limiting example, spleen cells and myeloma cells can bepelleted together. The pellet can then be slowly resuspended, over 30seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight1,450, Sigma) at 37° C. The fusion can then be stopped by slowly adding10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37° C.) over 1minute. The fused cells are centrifuged for 5 minutes at 500-1500 rpm.The cells are then resuspended in HAT medium (RPMI 1640 mediumcontaining 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodiumpyruvate, 4 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origen culturingsupplement (Fisher), 10% 653-conditioned RPMI 1640/Hepes media, 50 μM2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μMthymidine) and then plated at 200 μL/well in fifteen 96-well flat bottomtissue culture plates. The plates are then placed in a humidified 37° C.incubator containing 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-IL-12 Antibodies in Mouse Serum

Solid phase EIA's can be used to screen mouse sera for human IgGantibodies specific for human IL-12. Briefly, plates can be coated withIL-12 at 2 μg/mL in PBS overnight. After washing in 0.15M salinecontaining 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v)BSA in PBS, 200 μL/well for 1 hour at RT. Plates are used immediately orfrozen at −20° C. for future use. Mouse serum dilutions are incubated onthe IL-12 coated plates at 50 μL/well at RT for 1 hour. The plates arewashed and then probed with 50 μL/well HRP-labeled goat anti-human IgG,Fc specific diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT. The platescan again be washed and 100 μL/well of the citrate-phosphate substratesolution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H₂O₂ and 1mg/mL OPD) is added for 15 minutes at RT. Stop solution (4N sulfuricacid) is then added at 25 μL/well and the OD's are read at 490 nm via anautomated plate spectrophotometer.

Detection of Completely Human Immunoglobulins in Hybridoma Supernates

Growth positive hybridomas secreting fully human immunoglobulins can bedetected using a suitable EIA. Briefly, 96 well pop-out plates (VWR,610744) can be coated with 10 μg/mL goat anti-human IgG Fc in sodiumcarbonate buffer overnight at 4° C. The plates are washed and blockedwith 1% BSA-PBS for one hour at 37° C. and used immediately or frozen at−20° C. Undiluted hybridoma supernatants are incubated on the plates forone hour at 37° C. The plates are washed and probed with HRP labeledgoat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for one hour at 37°C. The plates are then incubated with substrate solution as describedabove.

Determination of Fully Human Anti-IL-12 Reactivity

Hybridomas, as above, can be simultaneously assayed for reactivity toIL-12 using a suitable RIA or other assay. For example, supernatants areincubated on goat anti-human IgG Fc plates as above, washed and thenprobed with radiolabled IL-12 with appropriate counts per well for 1hour at RT. The wells are washed twice with PBS and bound radiolabledIL-12 is quantitated using a suitable counter.

Human IgG1κ anti-IL-12 secreting hybridomas can be expanded in cellculture and serially subcloned by limiting dilution. The resultingclonal populations can be expanded and cryopreserved in freezing medium(95% FBS, 5% DMSO) and stored in liquid nitrogen.

Isotyping

Isotype determination of the antibodies can be accomplished using an EIAin a format similar to that used to screen the mouse immune sera forspecific titers. IL-12 can be coated on 96-well plates as describedabove and purified antibody at 2 μg/mL can be incubated on the plate forone hour at RT. The plate is washed and probed with HRP labeled goatanti-human IgG₁ or HRP labeled goat anti-human IgG₃ diluted at 1:4000 in1% BSA-PBS for one hour at RT. The plate is again washed and incubatedwith substrate solution as described above.

Binding Kinetics of Human Anti-Human IL-12 Antibodies with Human IL-12

Binding characteristics for antibodies can be suitably assessed using anIL-12 capture EIA and BIAcore technology, for example. Gradedconcentrations of purified human IL-12 antibodies can be assessed forbinding to EIA plates coated with 2 μg/mL of IL-12 in assays asdescribed above. The OD's can be then presented as semi-log plotsshowing relative binding efficiencies.

Quantitative binding constants can be obtained, e.g., as follows, or byany other known suitable method. A BIAcore CM-5 (carboxymethyl) chip isplaced in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cellof the chip at 5 μL/minute until a stable baseline is obtained. Asolution (100 μL) of 15 mg of EDC(N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200μL water is added to 100 μL of a solution of 2.3 mg of NHS(N-hydroxysuccinimide) in 200 μL water. Forty (40) μL of the resultingsolution is injected onto the chip. Six μL of a solution of human IL-12(15 μg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip,resulting in an increase of ca. 500 RU. The buffer is changed to TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mMcalcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg/mLBSA, pH 7.4) and flowed over the chip overnight to equilibrate it and tohydrolyze or cap any unreacted succinimide esters.

Antibodies are dissolved in the running buffer at 33.33, 16.67, 8.33,and 4.17 nM. The flow rate is adjusted to 30 μL/min and the instrumenttemperature to 25° C. Two flow cells are used for the kinetic runs, oneon which IL-12 had been immobilized (sample) and a second, underivatizedflow cell (blank). 120 μL of each antibody concentration is injectedover the flow cells at 30 μL/min (association phase) followed by anuninterrupted 360 seconds of buffer flow (dissociation phase). Thesurface of the chip is regenerated (interleukin-12/antibody complexdissociated) by two sequential injections of 30 μL each of 2 M guanidinethiocyanate.

Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, asknown in the art. For each antibody concentration the blank sensogram issubtracted from the sample sensogram. A global fit is done for bothdissociation (k_(d), sec⁻¹) and association (k_(a), mol⁻¹ sec⁻¹) and thedissociation constant (K_(D), mol) calculated (k_(d)/k_(a)). Where theantibody affinity is high enough that the RUs of antibody capturedare >100, additional dilutions of the antibody are run.

Results and Discussion

Generation of Anti-Human IL-12 Monoclonal Antibodies

Several fusions are performed and each fusion is seeded in 15 plates(1440 wells/fusion) that yield several dozen antibodies specific forhuman IL-12. Of these, some are found to consist of a combination ofhuman and mouse Ig chains. The remaining hybridomas secrete anti-IL-12antibodies consisting solely of human heavy and light chains. Of thehuman hybridomas, all are expected to be IgG1κ.

Binding Kinetics of Human Anti-Human IL-12 Antibodies

ELISA analysis confirms that purified antibody from most or all of thesehybridomas bind IL-12 in a concentration-dependent manner. FIGS. 1-2show the results of the relative binding efficiency of these antibodies.In this case, the avidity of the antibody for its cognate antigen(epitope) is measured. It should be noted that binding IL-12 directly tothe EIA plate can cause denaturation of the protein and the apparentbinding affinities cannot be reflective of binding to undenaturedprotein. Fifty percent binding is found over a range of concentrations.

Quantitative binding constants are obtained using BIAcore analysis ofthe human antibodies and reveals that several of the human monoclonalantibodies are very high affinity with K_(D) in the range of 1×10⁻⁹ to7×10⁻¹².

CONCLUSIONS

Several fusions are performed utilizing splenocytes from hybrid micecontaining human variable and constant region antibody transgenes thatare immunized with human IL-12. A set of several completely human IL-12reactive IgG monoclonal antibodies of the IgG1κ isotype are generated.The completely human anti-IL-12 antibodies are further characterized.Several of generated antibodies have affinity constants between 1×10⁹and 9×10¹². The unexpectedly high affinities of these fully humanmonoclonal antibodies make them suitable for therapeutic applications inIL-12-dependent diseases, pathologies or related conditions.

Example 3 C340 is a Neutralizing Human Monoclonal Antibody

The bioactivity of IL-12 was shown to be neutralized by C340 in avariety of IL-12 dependent activity assays. Since IL-12 enhances IFNGAMMA production by NK cells and T lymphocytes, the effect of C340antibody on the upregulation of IFN GAMMA mRNA and the effect of C340 onthe production of IFN GAMMA protein was examined (Trinchieri, G.,Current Opinion in Immunology, 9:17-23 (1997), Morris, S. C., et al.,Journal of Immunology, 152:1047-1056 (1994)). The ability of C340 toneutralize IL-12 driven induction of lymphokine activated killer (LAK)cell activity was also investigated in these studies (Kutza, J. andMurasko, D. M., Mechanisms of Ageing and Development, 90:209-222 (1996),Stern, A. S., et al., Proceedings of the National Academy of Sciences ofthe U.S.A., 87:6808-6812 (1990)). Lastly, the effect of C340 onIL-12-mediated upregulation of CD95 cell surface expression on T and NKcells was tested (Medvedev, A. E., et al., Cytokine, 9:394-404 (1997)).

Inhibition of IFN Gamma mRNA Transcription

To determine whether C340 inhibits IL-12/IL-2 induced IFN GAMMA genetranscription in human PBL, a reverse transcription-PCR assay wasperformed. Specific primers for β-actin (a control for mRNA integrityand content) and IFN GAMMA were used to amplify the cDNA obtained fromstimulated human PBL. FIG. 3 shows C340 down regulates IFN GAMMA mRNA inIL-12/IL-2 activated (2 hour) PBMC.

Inhibition of Intracellular IFN GAMMA as Measured by Flow Cytometry

In response to various signals and as a measure of activation, T cellsand NK cells can be induced to secrete cytokines. More specifically, PBLtreated with IL-2 and IL-12 initiate substantial synthesis of IFN gammawithin 4-8 hours after stimulation. This production can be detected inthe cytoplasm of Brefeldin-A treated PBL by flow cytometry. FIG. 4demonstrates a 60% reduction in IFN GAMMA production in such cultureswhen C340 IL-12 was added in conjunction with IL-12 for five hours.

Inhibition of IL-12 Induced IFN GAMMA Secretion

FIG. 5 clearly shows that two different lots of C340 inhibited thesecretion of IFN GAMMA by peripheral blood lymphocytes in adose-dependent fashion. Four hundred picograms of IL-12 were premixedwith varying amounts of C340 and then added to IL-2 stimulated culturesof PBL's. When IFN GAMMA was measured by EIA after an 18-24 hourincubation, markedly diminished amounts of IFN GAMMA were detected withas little as 1 μg/mL of C340 antibody.

Inhibition of IL-12 Induced LAK Cell Cytotoxicity

Raji cells, an IL-12 sensitive Burkitt lymphoma derived cell line, is anNK cell resistant, LAK cell sensitive cell line. Raji cells, intriplicate, were cultured for four hours with LAK cells which had beenactivated with 400 pg/mL IL-12 and 10 U/mL IL-2 in the presence orabsence of the human monoclonal antibody C340 (5000 ng/mL or 50 ng/mL).FIG. 6 shows the results from three normal, healthy donors. IL-12+IL-2activation of effector cells resulted in an increasing cytotoxicactivity over that of cells activated with IL-2 alone. The C340 antibodyinhibited this IL-12 dependent effect. The magnitude of inhibition wasrelated to antibody concentration, with the highest concentration testedreducing cytotoxicity to background levels.

Inhibition of CD95 Upregulation

Reports have described IL-12-induced upregulation of CD95 on the surfaceof highly purified CD56+ PBL. As can be seen in FIGS. 7A and 7B,distributional flow cytometric analysis revealed that CD95 expressionwas significantly upregulated on CD3+ T cells and CD56+ NK cells aftertreatment with IL-12 plus IL-2 for 72 hours. Concomitant anti-IL-12treatment inhibited CD95 expression in both CD3+ and CD56+ populations.CD3+ cells were inhibited by ˜50% (FIG. 7A), whereas CD56+ cells wereinhibited by ˜85% (FIG. 7B), as evidenced by a diminished MFI index(percent greater then unstimulated control).

Example 4 Gene Cloning and Characterization

Genomic DNA fragments containing either the C340 heavy chain gene or theC340 light chain were cloned and purified. Genomic DNA purified fromC340 hybridoma cells was partially digested with Sau3A restrictionenzyme and size-selected by centrifugal fractionation through a 10-40%sucrose gradient. DNA fragments in the size range of 15-23 kb werecloned into the bacteriophage vector, EMBL3, and packaged into phageparticles. Several packaging reactions resulted in a library of 1million bacteriophage clones. Approximately 600,000 clones from thelibrary were screened by plaque hybridization using 32P-labeled genomicDNA fragments that contained either human IgG1 heavy chain constantregion sequences or human kappa light chain constant region sequences asprobe. Thirteen heavy chain and nine light chain clones were detected.Of these, three heavy chain clones and four light chain clones werepurified by two more rounds of screening. One of the heavy chain clonesand two of the light chain clones were shown to contain the 5′ and 3′ends of the coding sequences by PCR analysis of bacteriophage DNA. TheDNA insert in heavy chain (HC) clone H4 was 16 kb in size and includes3.6 kb of 5′ flanking and at least 2 kb of 3′ flanking sequence. The DNAinsert in light chain (LC) clone LC1 was 15 kb in size and included 4.4kb of 5′ flanking and 6.0 kb of 3′ flanking sequence. The completeinserts were removed from the bacteriophage vector as SalI fragments andcloned between the XhoI and SalI sites of plasmid expression vectorp1351, which provided a gpt selectable marker gene. Because there was aninternal SalI site in the heavy chain variable region coding sequence,two SalI fragments had to be transferred from bacteriophage H4 to thep1351 expression vector. The resulting heavy and light chain expressionplasmids were termed p1560 and p1558, respectively. The orientations ofthe heavy and light chain genes in these two plasmids relative to thep1351 vector sequences were determined using restriction enzyme analysisand PCR, respectively. In both cases, the orientations were such thatthe 5′ end of the Ab gene fragment was proximal to the 3′ end of the gptgene. Both strands of the coding regions of the cloned genes weresequenced. The sequences of plasmids p1560 and p1558 are presented inFIGS. 11A-11K and FIGS. 13A-13J, respectively.

Example 5 Preparation of Recombinant Cell Lines

Heavy chain plasmid p1560 was linearized by digestion with PvuIrestriction enzyme and light chain plasmid p1558 was linearized usingSalI restriction enzyme. p3×63Ag8.653 (653) and SP2/0-Ag14 (SP2/0) cellswere separately transfected with the premixed linearized plasmids byelectroporation and cells cultured and transfectants selected usingmycophenolic acid as described (Knight, et al., Molecular Immunology30:1443 (1993)). Cell supernatants from mycophenolic acid-resistantcolonies were assayed approximately two weeks later for human IgG (i.e.,recombinant C340 (rC340)). For this, cell supernatants were incubated on96-well ELISA plates that were coated with goat antibodies specific forthe Fc portion of human IgG. Human IgG which bound to the coated platewas detected using alkaline phosphatase-conjugated goat anti-human IgG(heavy chain+light chain) antibody and alkaline phosphatase substratesas described (Knight, et al., Molecular Immunology 30:1443 (1993)).Cells of the higher producing clones were transferred to 24-well culturedishes in standard media and expanded (IMDM, 5% FBS, 2 mM glutamine,mycophenolic acid selection mix). The amount of antibody produced (i.e.,secreated into the media of spent cultures) was carefully quantified byELISA using purified C340 mAb as the standard. Selected clones were thenexpanded in T75 flasks and the production of human IgG by these cloneswas quantified by ELISA. Based on these values, six independent 653transfectants and three independent SP2/0 transfectants were subcloned(by seeding an average of one cell per well in 96 well plates), thequantity of antibody produced by the subclones was determined byassaying (ELISA) supernatants from individual subclone colonies. Threesubclones, 653 transfectant 19-20 (C379B) and the SP2/0 transfectants84-81 (C381A) and 22-56 (C389A), were selected for further analysis.

Assay for rC340 Antigen Binding

Prior to subcloning selected cell lines as described above, cellsupernatants from three parental lines (653 transfectants clone 2 andclone 18 and SP2/0 transfectant clone 1) were used to test the antigenbinding characteristics of rC340. The concentrations of rC340 in thethree cell supernatant samples were first determined by ELISA. Titratingamounts of the supernatant samples, or purified C340 positive control,were then incubated in 96-well plates coated with 2 μg/ml of humanIL-12. Bound mAb was then detected with alkaline phosphatase-conjugatedgoat anti-human IgG (heavy chain+light chain) antibody and theappropriate alkaline phosphatase substrates. As shown in FIG. 8, rC340bound specifically to human IL-12 in a manner indistinguishable from theoriginal C340 mAb.

Characterization of Selected Cell Lines

Growth curve analyses were performed on C379B, C381A, and C389A byseeding T75 flasks with a starting cell density of 2×105 cells/ml instandard media or SFM-5 serum-free media and then monitoring cell numberand rC340 concentration on a daily basis until the cultures were spent.The results of cultures in standard media are shown in FIGS. 9A-9C.Maximal C340 mAb production levels for C379B, C381A, and C389A were 135μg/ml, 150 μg/ml, and 110 μg/ml, respectively. Attempts to adapt C379Bcells to SFM-5 media were not successful. C381A cells produced the sameamount of rC340 in SFM-5 media as in standard media, whereas C389A cellsproduced only half as much rC340 in SFM-5 media as in standard media.

The stability of rC340 mAb production over time for the three subcloneswas assessed by culturing cells in 24-well dishes with standard media orstandard media without mycophenolic acid selection for varying periodsof time. Lines C379B and C381A were observed to stably produce rC340 inthe presence or absence of selection for a period of 30 days (themaximum time tested) and 75 days, respectively. Line C389A was unstableand after 43 days of culture produced just 20% as much antibody as atthe beginning of the study.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

1. An isolated nucleic acid molecule encoding an anti-IL-12 antibody,comprising a polynucleotide encoding a heavy chain complementaritydetermining region 1 (CDR1) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:1, a polynucleotide encoding a heavy chain complementaritydetermining region 2 (CDR2) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:2, a polynucleotide encoding a heavy chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:3, a polynucleotide encoding a light chain complementaritydetermining region 1 (CDR1) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:4, a polynucleotide encoding a light chain complementaritydetermining region 2 (CDR2) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:5, and a polynucleotide encoding a light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence with notmore than one conservative substitution from the amino acid sequence ofSEQ ID NO:6, wherein the antibody is a variant of the antibodycomprising the heavy chain CDR1 amino acid sequence of SEQ ID NO:1, theheavy chain CDR2 amino acid sequence of SEQ ID NO:2, the heavy chainCDR3 amino acid sequence of SEQ ID NO:3, the light chain CDR1 amino acidsequence of SEQ ID NO:4, the light chain CDR2 amino acid sequence of SEQID NO:5, and the light chain CDR3 amino acid sequence of SEQ ID NO:6. 2.The isolated nucleic acid molecule according to claim 1, wherein theantibody is human.
 3. An isolated nucleic acid vector comprising theisolated nucleic acid molecule according to claim
 2. 4. An isolated hostcell comprising the isolated nucleic vector according to claim 3,wherein the host cell is prokaryotic or eukaryotic.
 5. The host cellaccording to claim 4, wherein said host cell is at least one selectedfrom COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 293, HeLa,P3X63Ag8.653, SP2/0-Ag14, mouse NIH3T3, C127, CV-1, quail QC1-3, mouseL, H9, and Jurkat cells.
 6. A method for producing an anti-IL-12antibody, comprising culturing the isolated host cell of claim 4 underconditions such that the IL-12 antibody is expressed and recovered.
 7. Acomposition comprising the isolated nucleic acid molecule according toclaim 1, and at least one pharmaceutically acceptable carrier ordiluent.
 8. An isolated nucleic acid molecule encoding an anti-IL-12antibody, comprising a polynucleotide encoding a heavy chain CDR1comprising the amino acid sequence of SEQ ID NO:1, a polynucleotideencoding a heavy chain CDR2 comprising the amino acid sequence of SEQ IDNO:2, a polynucleotide encoding a heavy chain CDR3 comprising the aminoacid sequence with not more than one conservative substitution from theamino acid sequence of SEQ ID NO:3, a polynucleotide encoding a lightchain CDR1 comprising the amino acid sequence of SEQ ID NO:4, apolynucleotide encoding a light chain CDR2 comprising the amino acidsequence of SEQ ID NO:5, and a polynucleotide encoding a light chainCDR3 comprising the amino acid sequence with not more than oneconservative substitution from the amino acid sequence of SEQ ID NO:6.9. The isolated nucleic acid molecule according to claim 8, wherein theantibody is human.
 10. An isolated nucleic acid vector comprising theisolated nucleic acid molecule according to claim
 9. 11. An isolatedhost cell comprising the isolated nucleic vector according to claim 10,wherein the host cell is prokaryotic or eukaryotic.
 12. The host cellaccording to claim 11, wherein said host cell is at least one selectedfrom COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 293, HeLa,P3X63Ag8.653, SP2/0-Ag14, mouse NIH3T3, C127, CV-1, quail QC1-3, mouseL, H9, and Jurkat cells.
 13. A method for producing an anti-IL-12antibody, comprising culturing the isolated host cell of claim 11 underconditions such that the IL-12 antibody is expressed and recovered. 14.A composition comprising the isolated nucleic acid molecule according toclaim 8, and at least one pharmaceutically acceptable carrier ordiluent.